WNT1 mutations in early-onset osteoporosis and osteogenesis imperfecta.

This report identifies human skeletal diseases associated with mutations in WNT1. In 10 family members with dominantly inherited, early-onset osteoporosis, we identified a heterozygous missense mutation in WNT1, c.652T→G (p.Cys218Gly). In a separate family with 2 siblings affected by recessive osteogenesis imperfecta, we identified a homozygous nonsense mutation, c.884C→A, p.Ser295*. In vitro, aberrant forms of the WNT1 protein showed impaired capacity to induce canonical WNT signaling, their target genes, and mineralization. In mice, Wnt1 was clearly expressed in bone marrow, especially in B-cell lineage and hematopoietic progenitors; lineage tracing identified the expression of the gene in a subset of osteocytes, suggesting the presence of altered cross-talk in WNT signaling between the hematopoietic and osteoblastic lineage cells in these diseases.

Rare variations in WNT3A and DKK1 may predispose carriers to primary osteoporosis.

Childhood-onset primary osteoporosis is manifested as reduced bone mineral density, peripheral fractures and/or vertebral compression fractures. Until now, only mutations in LRP5 have been shown to cause the disorder. Candidate gene analyses were performed on 15 patients with primary osteoporosis and 80 healthy controls using CSGE and sequencing. The genes studied included DKK1, DKK2, WNT3A, WNT10B, AXIN1, SOST, TPH1 and 5-HTR1B. Two rare variants in WNT3A (c.152A > G, p.K51R) and DKK1 (c.359G > T, p.R120L) were identified in two patients and their affected family members, but not in control subjects, suggesting a significance for the skeletal phenotype. The in vitro studies of variants showed reduced signaling activity in p.K51R-Wnt3a, while no differences were observed between the WT and variant forms of DKK1. This study addresses the role of other components of the canonical Wnt signaling pathway besides LRP5 in primary osteoporosis, and putatively associates WNT3A and DKK1 variants with the disorder. Future functional studies are needed to elucidate the functional effects of the variants.

Primary osteoporosis without features of OI in children and adolescents: clinical and genetic characteristics.

Our aim was to characterize clinical findings and familial associations, and to examine candidate genes for disease-causing mutations in a cohort of children suffering from primary osteoporosis without features of osteogenesis imperfecta. Patients with osteoporosis and their nuclear families were studied. Medical history was reviewed. Calcium homeostasis parameters were measured and spinal radiographs obtained. BMD was determined by DXA for patients, parents and siblings. LRP5, LRP6, and PTHLH genes were sequenced. Twenty-seven patients (14 males) from 24 families were recruited. Median age at presentation was 10.1 years (range 3.3-15.6 years). One-third of the children had at least one parent with a BMD below the expected range for age. LRP5, LRP6, and PTHLH showed no causative mutations. Four polymorphisms in LRP5 were overrepresented in patients; the minor allele frequency of Q89R, V667M, N740N, and A1330V was significantly higher than in controls. Age of onset, clinical severity, and inheritance patterns are variable in children with primary osteoporosis. Several patients had evidence suggestive of familial transmission. The underlying genetic factors remain to be elucidated.

Mutations in LRP5 cause primary osteoporosis without features of OI by reducing Wnt signaling activity.

BACKGROUND: Primary osteoporosis is a rare childhood-onset skeletal condition whose pathogenesis has been largely unknown. We have previously shown that primary osteoporosis can be caused by heterozygous missense mutations in the Low-density lipoprotein receptor-related protein 5 (LRP5) gene, and the role of LRP5 is further investigated here. METHODS: LRP5 was analyzed in 18 otherwise healthy children and adolescents who had evidence of osteoporosis (manifested as reduced bone mineral density i.e. BMD, recurrent peripheral fractures and/or vertebral compression fractures) but who lacked the clinical features of osteogenesis imperfecta (OI) or other known syndromes linked to low BMD. Also 51 controls were analyzed. Methods used in the genetic analyses included direct sequencing and multiplex ligation-dependent probe amplification (MLPA). In vitro studies were performed using luciferase assay and quantitative real-time polymerase chain reaction (qPCR) to examine the effect of two novel and three previously identified mutations on the activity of canonical Wnt signaling and on expression of tryptophan hydroxylase 1 (Tph1) and 5-hydroxytryptamine (5-Htr1b). RESULTS: Two novel LRP5 mutations (c.3446 T > A; p.L1149Q and c.3553 G > A; p.G1185R) were identified in two patients and their affected family members. In vitro analyses showed that one of these novel mutations together with two previously reported mutations (p.C913fs, p.R1036Q) significantly reduced the activity of the canonical Wnt signaling pathway. Such reductions may lead to decreased bone formation, and could explain the bone phenotype. Gut-derived Lrp5 has been shown to regulate serotonin synthesis by controlling the production of serotonin rate-limiting enzyme, Tph1. LRP5 mutations did not affect Tph1 expression, and only one mutant (p.L1149Q) reduced expression of serotonin receptor 5-Htr1b (p < 0.002). CONCLUSIONS: Our results provide additional information on the role of LRP5 mutations and their effects on the development of juvenile-onset primary osteoporosis, and hence the pathogenesis of the disorder. The mutations causing primary osteoporosis reduce the signaling activity of the canonical Wnt signaling pathway and may therefore result in decreased bone formation. The specific mechanism affecting signaling activity remains to be resolved in future studies.

How stand productivity results from size- and competition-dependent growth and mortality.

BACKGROUND: A better understanding of the relationship between stand structure and productivity is required for the development of: a) scalable models that can accurately predict growth and yield dynamics for the world's forests; and b) stand management regimes that maximize wood and/or timber yield, while maintaining structural and species diversity. METHODS: We develop a cohort-based canopy competition model ("CAIN"), parameterized with inventory data from Ontario, Canada, to examine the relationship between stand structure and productivity. Tree growth, mortality and recruitment are quantified as functions of diameter and asymmetric competition, using a competition index (CAI(h)) defined as the total projected area of tree crowns at a given tree's mid-crown height. Stand growth, mortality, and yield are simulated for inventoried stands, and also for hypothetical stands differing in total volume and tree size distribution. RESULTS: For a given diameter, tree growth decreases as CAI(h) increases, whereas the probability of mortality increases. For a given CAI(h), diameter growth exhibits a humped pattern with respect to diameter, whereas mortality exhibits a U-shaped pattern reflecting senescence of large trees. For a fixed size distribution, stand growth increases asymptotically with total density, whereas mortality increases monotonically. Thus, net productivity peaks at an intermediate volume of 100-150 m(3)/ha, and approaches zero at 250 m(3)/ha. However, for a fixed stand volume, mortality due to senescence decreases if the proportion of large trees decreases as overall density increases. This size-related reduction in mortality offsets the density-related increase in mortality, resulting in a 40% increase in yield. CONCLUSIONS: Size-related variation in growth and mortality exerts a profound influence on the relationship between stand structure and productivity. Dense stands dominated by small trees yield more wood than stands dominated by fewer large trees, because the relative growth rate of small trees is higher, and because they are less likely to die.

Mutations in TRPV4 cause an inherited arthropathy of hands and feet.

Familial digital arthropathy-brachydactyly (FDAB) is a dominantly inherited condition that is characterized by aggressive osteoarthropathy of the fingers and toes and consequent shortening of the middle and distal phalanges. Here we show in three unrelated families that FDAB is caused by mutations encoding p.Gly270Val, p.Arg271Pro and p.Phe273Leu substitutions in the intracellular ankyrin-repeat domain of the cation channel TRPV4. Functional testing of mutant TRPV4 in HEK-293 cells showed that the mutant proteins have poor cell-surface localization. Calcium influx in response to the synthetic TRPV4 agonists GSK1016790A and 4αPDD was significantly reduced, and mutant channels did not respond to hypotonic stress. Others have shown that gain-of-function TRPV4 mutations cause skeletal dysplasias and peripheral neuropathies. Our data indicate that TRPV4 mutations that reduce channel activity cause a third phenotype, inherited osteoarthropathy, and show the importance of TRPV4 activity in articular cartilage homeostasis. Our data raise the possibility that TRPV4 may also have a role in age- or injury-related osteoarthritis.

Unicameral bone cysts: comparison of percutaneous curettage, steroid, and autologous bone marrow injections.

BACKGROUND: The purpose of this study was to compare the outcome of percutaneous curettage with intralesional injection of methylprednisolone and bone marrow for unicameral bone cysts (UBCs). METHODS: This was a retrospective review of 46 children and adolescents with UBC treated with autologous bone marrow injection, methylprednisolone acetate injection or percutaneous curettage alone. Inclusion criteria were a radiological diagnosis of UBC and at least 24 months follow-up from the last procedure. Healing was determined using Neer/Cole 4-grades rating scale. RESULTS: The 3 treatment groups were comparable with regard to age, sex, location of the cyst, and the number of procedures undertaken. At 2 years follow-up, the proportion of patients with satisfactory healing (Neer/Cole grades I and II) was greatest among those who underwent percutaneous curettage (70%) compared with bone marrow injection (21%) and methylprednisolone acetate injection (41%) (P = 0.03). We found no association between healing and age (P = 0.80) nor between healing and sex (P = 0.61). CONCLUSIONS: These results suggest that mechanical disruption of the cyst membrane may be helpful in healing of cysts and that this technique may be preferred to simple intralesional injections. LEVEL OF EVIDENCE: Level III.

Early-onset metaphyseal chondrodysplasia type Schmid associated with a COL10A1 frame-shift mutation and impaired trimerization of wild-type α1(X) protein chains.

Both dominant-negative and haploinsufficiency effects have been proposed in the pathogenesis of metaphyseal chondrodysplasia type Schmid (MCDS) due to nonsense and frame-shift mutations of COL10A1. This study examines these alternative effects. A proband with typical early-onset MCDS was ascertained and COL10A1 sequencing undertaken. The assembly of trimeric collagen X molecules was studied using in vitro coupled transcription and translation of wild-type and mutant α1(X) cDNAs. The proband was heterozygous for a unique COL10A1 mutation, c.1735_1739del5ins22. Mutant protein chains, with the corresponding p.G579fsX611 change, failed to spontaneously trimerize. When wild-type α1(X) chains were translated alone, 57 ± 7% of the chains assembled into stable collagen X trimers. Trimerization of wild-type chains was significantly reduced to 33 ± 6% when translated in 1:1 mixtures with p.G579fsX611 α1(X) chains. The protein assembly assay showed that the mutant chains exerted a dominant-negative effect on collagen X assembly. Previous studies indicate that nonsense-mediated decay, activation of endoplasmic reticulum, and unfolded protein responses as well as altered chondrocyte differentiation are the major determinants of phenotypic severity and age of presentation. We speculate that complete loss of mutant transcripts yields COL10A1 haploinsufficiency and late clinical presentation while incomplete loss of mutant transcripts yields dominant-negative effects with early clinical presentation.

Long-term clinical outcome and carrier phenotype in autosomal recessive hypophosphatemia caused by a novel DMP1 mutation.

Homozygous inactivating mutations in DMP1 (dentin matrix protein 1), the gene encoding a noncollagenous bone matrix protein expressed in osteoblasts and osteocytes, cause autosomal recessive hypophosphatemia (ARHP). Herein we describe a family with ARHP owing to a novel homozygous DMP1 mutation and provide a detailed description of the associated skeletal dysplasia and carrier phenotype. The two adult patients with ARHP, a 78-year-old man and his 66-year-old sister, have suffered from bone pain and lower extremity varus deformities since early childhood. With increasing age, both patients developed severe joint pain, contractures, and complete immobilization of the spine. Radiographs showed short and deformed long bones, significant cranial hyperostosis, enthesopathies, and calcifications of the paraspinal ligaments. Biochemistries were consistent with hypophosphatemia owing to renal phosphate wasting; markers of bone turnover and serum fibroblast growth factor 23 (FGF-23) levels were increased significantly. Nucleotide sequence analysis of DMP1 revealed a novel homozygous mutation at the splice acceptor junction of exon 6 (IVS5-1G > A). Two heterozygous carriers of the mutation also showed mild hypophosphatemia, and bone biopsy in one of these individuals showed focal areas of osteomalacia. In bone, DMP1 expression was absent in the homozygote but normal in the heterozygote, whereas FGF-23 expression was increased in both subjects but higher in the ARHP patient. The clinical and laboratory observations in this family confirm that DMP1 has an important role in normal skeletal development and mineral homeostasis. The skeletal phenotype in ARHP may be significantly more severe than in other forms of hypophosphatemic rickets.

Characterization of the six zebrafish clade B fibrillar procollagen genes, with evidence for evolutionarily conserved alternative splicing within the pro-alpha1(V) C-propeptide.

Genes for tetrapod fibrillar procollagen chains can be divided into two clades, A and B, based on sequence homologies and differences in protein domain and gene structures. Although the major fibrillar collagen types I-III comprise only clade A chains, the minor fibrillar collagen types V and XI comprise both clade A chains and the clade B chains pro-alpha1(V), pro-alpha3(V), pro-alpha1(XI) and pro-alpha2(XI), in which defects can underlie various genetic connective tissue disorders. Here we characterize the clade B procollagen chains of zebrafish. We demonstrate that in contrast to the four tetrapod clade B chains, zebrafish have six clade B chains, designated here as pro-alpha1(V), pro-alpha3(V)a and b, pro-alpha1(XI)a and b, and pro-alpha2(XI), based on synteny, sequence homologies, and features of protein domain and gene structures. Spatiotemporal expression patterns are described, as are conserved and non-conserved features that provide insights into the function and evolution of the clade B chain types. Such features include differential alternative splicing of NH(2)-terminal globular sequences and the first case of a non-triple helical imperfection in the COL1 domain of a clade B, or clade A, fibrillar procollagen chain. Evidence is also provided for previously unknown and evolutionarily conserved alternative splicing within the pro-alpha1(V) C-propeptide, which may affect selectivity of collagen type V/XI chain associations in species ranging from zebrafish to human. Data presented herein provide insights into the nature of clade B procollagen chains and should facilitate their study in the zebrafish model system.

Low density lipoprotein receptor-related protein 5 (LRP5) mutations and osteoporosis, impaired glucose metabolism and hypercholesterolaemia.

OBJECTIVE: Mutations in the low-density lipoprotein receptor-related protein 5 gene (LRP5) underlie osteoporosis-pseudoglioma syndrome. Animal models implicate a role for LRP5 in lipid and glucose homeostasis. The objective was to evaluate metabolic consequences of LRP5 mutations in humans. DESIGN AND PATIENTS: Thirteen Finnish individuals with homozygous or heterozygous LRP5 mutations were assessed for bone health, glucose and lipid metabolism, and for serum serotonin concentration. Results were compared with findings in family members without mutations. MEASUREMENTS: Bone mineral density (BMD), vertebral morphology, oral and intravenous glucose tolerance tests, lipid profile and serum serotonin concentrations. RESULTS: Two individuals were homozygous for R570W, one compound heterozygous for R570W and R1036Q, and 10 were heterozygous (six for R570W, three for R1036Q and one for R925C). Subjects with two LRP5 mutations had multiple spinal fractures and low BMD. Subjects with one mutation had significantly lower median lumbar spine (P = 0.004) and femoral neck (P = 0.005) BMD Z-scores, and more often vertebral fractures than the 18 individuals without mutations. Of the 12 subjects with LRP5 mutation six had diabetes and one had impaired glucose tolerance. Intravenous glucose tolerance tests suggested impaired beta-cell function; no insulin resistance was observed. Prevalence of hypercholesterolaemia was similar in mutation positive and negative subjects. Serum serotonin concentrations showed a trend towards higher concentrations in subjects with LRP5 mutation. CONCLUSIONS: We found high prevalence of osteoporosis and abnormal glucose metabolism in subjects with LRP5 mutation(s). Further studies are needed to establish the role of LRP5 in glucose and lipid metabolism.

Early-onset osteoarthritis due to otospondylomegaepiphyseal dysplasia in a family with a novel splicing mutation of the COL11A2 gene.

OBJECTIVE: To evaluate an approach to the clinical, radiographic, and molecular diagnosis of an underlying skeletal dysplasia in adults presenting with early-onset polyarticular osteoarthritis (OA). METHODS: We identified a family with 2 adults with polyarticular OA and a child with generalized arthralgia. General, musculoskeletal, ocular, and auditory evaluations were undertaken. Investigations included radiographs of symptomatic joints, analysis of serum inflammatory markers and joint fluid, and mutational analyses of the COL11A2 gene. RESULTS: The 3 affected individuals had normal stature, mild mid-face hypoplasia, and hearing impairment, but normal eyes. Radiographs of the affected adults showed severe polyarticular OA but did not reveal diagnostic evidence of an underlying skeletal dysplasia. However, the child's radiographs showed enlarged epiphyses with an advanced bone age. The combination of skeletal, facial, and auditory features together with the absence of ocular features indicated that they had otospondylomegaepiphyseal dysplasia, also known as Stickler syndrome type III. The diagnosis was confirmed by identifying a mutation in the COL11A2 gene that encodes the pre-pro-alpha2(XI) chain of type XI collagen that is involved in type II collagen fibrillogenesis. CONCLUSION: Early-onset polyarticular OA may occur in adults without a known or obvious underlying skeletal dysplasia. This study provides an approach to the diagnosis of an underlying skeletal dysplasia in such individuals.

Shwachman-Diamond syndrome is associated with low-turnover osteoporosis.

INTRODUCTION: Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder characterized by exocrine pancreatic insufficiency and bone marrow dysfunction. These result in malabsorption and hematological abnormalities. A skeletal dysplasia is also an integral feature of SDS. The present study assessed prevalence and determinants of osteopenia and osteoporosis in patients with SDS and disease-causing mutations in the SBDS gene. MATERIALS AND METHODS: Eleven patients (8 males) aged from 5 to 37 years (median 16.7 years) with a genetically confirmed diagnosis of SDS were assessed for fracture history, bone mineral content (BMC), lean tissue mass (LTM) and bone mineral density (BMD) (Hologic Discovery A), osteoporotic vertebral changes, and for blood biochemistry and hematological parameters. Iliac crest bone biopsies were obtained from four patients for histology and histomorphometry. RESULTS: The main findings were: (1) markedly reduced BMD Z-scores at the lumbar spine (median -2.1, range -4.4 to -0.8), proximal femur (median -1.3, range -2.2 to -0.7) and, whole body (median -1.0, range -2.8 to +0.6), and reduced Z-scores for height-adjusted BMC/LTM ratio (median -0.9, range -3.6 to +1.1); (2) vertebral compression fractures in three patients; and (3) blood biochemistry suggestive of mild vitamin D and vitamin K deficiency. Bone biopsies in four patients showed significant low-turnover osteoporosis with reduced trabecular bone volume, low numbers of osteoclasts and osteoblasts, and reduced amount of osteoid. CONCLUSIONS: The results suggest that in addition to the skeletal dysplasia, SDS is associated with a more generalized bone disease characterized by low bone mass, low bone turnover and by vertebral fragility fractures. Osteoporosis may result from a primary defect in bone metabolism, and could be related to the bone marrow dysfunction and neutropenia.

Treatment of open femur fractures in children: comparison between external fixator and intramedullary nailing.

BACKGROUND: Open femur fractures in children are uncommon and usually associated with other injuries. In adults, there is a current trend to treat open fractures with intramedullary (IM) devices. The goal of this study was to compare external fixator (EF) to IM devices in the treatment of open femur fractures in children. METHODS: Diaphyseal femur fractures without growth plate involvement were included. Thirty-five patients (12 IM; 23 EF) were identified. Age, hospital stay, polytrauma, mechanism of injury, and Gustilo-Anderson grade were recorded. Follow-up was at least until the fracture was clinically and radiographically healed. RESULTS: Patients with EFs were 5.2 times more likely (95% confidence interval, 1.05-25.5) to have any complication. Excluding pin track infections, patients with EFs were 2.7 times as likely (95% confidence interval, 0.567-13.2) to have a complication. Refractures occurred only in the EF group (6/23, 26%) and not in the IM nailing group (P = 0.062, Fischer exact test). These were associated with varus malunions-all 3 of the EF group with more than 15 degrees of varus at fracture union suffered a refracture. CONCLUSIONS: Treatment of open femur fractures in children is a challenging problem. Treatment with IM devices had fewer complications than the EF. We think that whenever possible, the use of IM devices for the treatment of open femur fracture in children should be considered, especially grade 1 open injuries. If EFs are used, avoiding varus malunion may decrease the refracture rate, and secondary change to an IM device should be considered. LEVEL OF EVIDENCE: Comparative cohort study. Grade 3 level of evidence.

COL10A1 nonsense and frame-shift mutations have a gain-of-function effect on the growth plate in human and mouse metaphyseal chondrodysplasia type Schmid.

Missense, nonsense and frame-shift mutations in the collagen X gene (COL10A1) result in metaphyseal chondrodysplasia type Schmid (MCDS). Complete degradation of mutant COL10A1 mRNA by nonsense-mediated decay in human MCDS cartilage implicates haploinsufficiency in the pathogenesis for nonsense mutations in vivo. However, the mechanism is unclear in situations where the mutant mRNA persist. We show that nonsense/frame-shift mutations can elicit a gain-of-function effect, affecting chondrocyte differentiation in the growth plate. In an MCDS proband, heterozygous for a p.Y663X nonsense mutation, the growth plate cartilage contained 64% wild-type and 36% mutant mRNA and the hypertrophic zone was disorganized and expanded. The in vitro translated mutant collagen X chains, which are truncated, were misfolded, unable to assemble into trimers and interfered with the assembly of normal alpha1(X) chains into trimers. Unlike Col10a1 null mutants, transgenic mice (FCdel) bearing the mouse equivalent of a human MCDS p.P620fsX621 mutation, displayed typical characteristics of MCDS with disproportionate shortening of limbs and early onset coxa vara. In FCdel mice, the degree of expansion of the hypertrophic zones was transgene-dosage dependent, being most severe in mice homozygous for the transgene. Chondrocytes in the lower region of the expanded hypertrophic zone expressed markers uncharacteristic of hypertrophic chondrocytes, indicating that differentiation was disrupted. Misfolded FCdel alpha1(X) chains were retained within the endoplasmic reticulum of hypertrophic chondrocytes, activating the unfolded protein response. Our findings provide strong in vivo evidence for a gain-of-function effect that is linked to the activation of endoplasmic reticulum-stress response and altered chondrocyte differentiation, as a possible molecular pathogenesis for MCDS.

Factors predicting the outcome of primary clubfoot surgery.

BACKGROUND: We aimed to determine the rate of further surgery, the functional outcome and the factors associated with outcome after primary clubfoot surgery. METHOD: We conducted a retrospective study of a cohort of all children who were less than 2 years of age at the time of surgery for idiopathic clubfoot deformity at the Hospital for Sick Children, Toronto, Ont., a tertiary care pediatric hospital. Of the 91 families who could be contacted, 63 agreed to return. The children's charts were reviewed, and their feet were given a Functional Rating System (FRS) score. RESULTS: Of the original operated population (n = 126), 75% were male and 41% had bilateral clubfoot. The average age at the time of surgery was 8 months, and the mean follow-up was 80.6 months. Further surgery was performed in 19% of cases. The mean FRS outcome score was 79. On average, the FRS score increased by 1.9 points as age at the time of surgery increased by 1 month. Only the presurgical talocalcaneal index was associated with the need for further surgery. CONCLUSION: The need for further surgery was 19% overall. Children who had surgery closer to 12 months of age had better functional results. Therefore, surgery should probably be performed in the second, rather than the first, 6 months of life.

HOXD10 M319K mutation in a family with isolated congenital vertical talus.

Congenital vertical talus (CVT) is a primary dislocation of the talonavicular joint that often occurs in neuromusculoskeletal syndromes, but may also be seen as an isolated abnormality. Six families with isolated CVT were ascertained. DNA was isolated from 21 affected individuals and 17 unaffected individuals from these families, as well as from five sporadic patients with CVT. Variable expressivity was noted in three families, manifesting as clubfoot in three individuals. Genome-wide linkage analysis generated a maximum two-point logarithm of odds score on chromosome 2q with D2S1353 (Zmax = 1.43 at theta(max) = 0.1), 17 Mb from the HOXD gene cluster. DNA from one affected individual of each family was subjected to mutational analysis of the HOXD10 gene. A single missense mutation was identified (M319K, 956T > A) in the homeodomain recognition helix of the HOXD10 gene that segregated with disease in one large British family. This mutation was recently described in a family of Italian descent with CVT and Charcot-Marie-Tooth deformity HOXD10 gene mutations were not identified in any of the other families or sporadic patients with CVT, suggesting that genetic heterogeneity underlies this disorder.

Structural abnormalities of the cornea and lid resulting from collagen V mutations.

PURPOSE: Type V collagen forms heterotypic fibrils with type I collagen and accounts for 10% to 20% of corneal collagen. The purpose of this study was to define the ocular phenotype resulting from mutations in the type V collagen genes COL5A1 and COL5A2 and to study the pathogenesis of anomalies in a Col5a1-deficient mouse. METHODS: Seven patients with classic Ehlers-Danlos syndrome (EDS) due to COL5A1 haploinsufficiency and one with an exon-skipping mutation in COL5A2 underwent an ocular examination, corneal topography, pachymetry, and specular microscopy. A Col5a1-haploinsufficient mouse model of classic EDS was used for biochemical and immunochemical analyses of corneas. Light and electron microscopy were used to quantify stromal thickness, fibril density, fibril structure, and diameter. RESULTS: Five males and three females (mean age, 26 +/- 13.57 years; range, 11-52) were studied. All patients had "floppy eyelids." The corneas of all eyes were thinner (mean corneal thickness: 435.75 +/- 12.51 microm) when compared with control corneas (568.89 +/- 28.46 microm; P < 0.0001). In the Col5a1+/- mouse cornea, type V collagen content was reduced by approximately 49%, and stromal thickness was reduced by approximately 26%. Total collagen deposition in Col5a1(+/-) corneas also was reduced. Collagen fibril diameters were increased, but fibril density was decreased throughout the stroma at all developmental stages. CONCLUSIONS: In the eye, COL5A1 and COL5A2 mutations manifest as abnormally thin and steep corneas with floppy eyelids. Mechanisms involved in producing the latter anomalies probably involve altered regulation of collagen fibrillogenesis due to abnormalities in heterotypic type I/V collagen interactions similar to those observed in the Col5a1+/- mouse cornea.

Multiple epiphyseal dysplasia mutations in MATN3 cause misfolding of the A-domain and prevent secretion of mutant matrilin-3.

Multiple epiphyseal dysplasia (MED) is a relatively common skeletal dysplasia that can present in childhood with a variable phenotype of short stature and pain and stiffness in the large joints, and often progresses to early-onset osteoarthritis in adulthood. Mutations in the matrilin-3 gene (MATN3) have recently been shown to underlie some forms of autosomal dominant MED. To date all MED mutations in matrilin-3 cluster in the single A-domain, suggesting that they may disrupt the structure and/or function of this important domain. To determine the effects of MATN3 mutations on the structure and function of matrilin-3 we expressed both normal and mutant matrilin-3 in mammalian cells. Wild-type (wt) matrilin-3 was efficiently secreted into conditioned medium, whereas mutant matrilin-3 was retained and accumulated within the cell. Furthermore, when the mutant A-domains were examined individually, they existed primarily in an unfolded conformation. Co-immunoprecipitation experiments demonstrated that the mutant A-domains were specifically associated with ERp72, a chaperone protein known to be involved in mediating disulfide bond formation. Light microscopy of cartilage from an MED patient with a MATN3 mutation showed the presence of intracellular material within the chondrocytes, whilst the overall matrix appeared normal. On electron micrographs, the inclusions noted at the light microscopy level appeared to be dilated cisternae of rough endoplasmic reticulum and immunohistochemical analysis confirmed that the retained protein was matrilin-3. In summary, the data presented in this paper suggest that MED caused by MATN3 mutations is the result of an intracellular retention of the mutant protein.